WO2006056204A1 - Methode permettant de reticuler de l'acide hyaluronique avec du divinylsulfone - Google Patents

Methode permettant de reticuler de l'acide hyaluronique avec du divinylsulfone Download PDF

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WO2006056204A1
WO2006056204A1 PCT/DK2005/000753 DK2005000753W WO2006056204A1 WO 2006056204 A1 WO2006056204 A1 WO 2006056204A1 DK 2005000753 W DK2005000753 W DK 2005000753W WO 2006056204 A1 WO2006056204 A1 WO 2006056204A1
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hydrogel
range
dvs
hyaluronic acid
composition
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PCT/DK2005/000753
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English (en)
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WO2006056204A8 (fr
Inventor
Fanny Longin
Khadija Schwach-Abdellaoui
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Novozymes Biopolymer A/S
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Priority to CN2005800471013A priority Critical patent/CN101107270B/zh
Priority to US11/719,790 priority patent/US8481080B2/en
Priority to PL05804061T priority patent/PL1817347T3/pl
Priority to EP05804061.9A priority patent/EP1817347B1/fr
Priority to DK05804061.9T priority patent/DK1817347T3/en
Priority to ES05804061.9T priority patent/ES2636998T3/es
Publication of WO2006056204A1 publication Critical patent/WO2006056204A1/fr
Publication of WO2006056204A8 publication Critical patent/WO2006056204A8/fr
Priority to US13/912,894 priority patent/US20130338100A1/en
Priority to US14/591,522 priority patent/US20150190420A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/042—Gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • TITLE Method of cross-linking hyaluronic acid with divinylsulfone
  • the present invention relates to a process for the preparation of modified hyaluronic acid (HA), in particular cross-linked HA, for use in cosmetic, biomedical and pharmaceutical applications.
  • HA modified hyaluronic acid
  • Hyaluronic acid is a natural and linear carbohydrate polymer belonging to the class of the non-sulfated glycosaminoglycans. It is composed of beta-1 ,3- ⁇ /-acetyl glucosamine and beta-1 ,4-glucuronic acid repeating disaccharide units with a molecular weight (MW) up to 6 MDa.
  • HA is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis. HA may be extracted from natural tissues including the connective tissue of vertebrates, from the human umbilical cord and from cocks' combs. However, it is preferred today to prepare it by microbiological methods to minimize the potential risk of transferring infectious agents, and to increase product uniformity, quality and availability (WO 03/0175902, Novozymes).
  • HA HA-binding protein
  • HA is soluble in water at room temperature, i.e. about 20 0 C, it is rapidly degraded by hyaluronidase in the body, and it is difficult to process into biomaterials.
  • Cross- linking of HA has therefore been introduced in order to improve the physical and mechanical properties of HA and its in vivo residence time.
  • U.S. patent No. 4,582,865 (Biomatrix Inc.) describes the preparation of cross-linked gels of HA, alone or mixed with other hydrophilic polymers, using divinyl sulfone (DVS) as the cross-linking agent.
  • the preparation of a cross-linked HA or salt thereof using a polyfunctional epoxy compound is disclosed in EP 0 161 887 B1.
  • Other bi- or poly-functional reagents that have been employed to cross-link HA through covalent linkages include formaldehyde (U.S.
  • a problem to be solved by the present invention is how to manufacture hyaluronic acid based hydrogels with improved properties, such as higher homogeneity, increased softness, and/or easier syringeability.
  • the cross-linked gels produced by the method of the invention show an increased homogeneity and an increased softness compared to the standard DVS crosslinked HA- hydrogels.
  • the gels resulting from the method of the instant invention are also easier to inject through a syringe, as shown in the examples.
  • the invention relates to a method of producing a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with divinylsulfone (DVS), said method comprising the steps of: (a) providing an alkaline solution of hyaluronic acid, or salt thereof;
  • step (b) adding DVS to the solution of step (a), whereby the hyaluronic acid, or salt thereof, is crosslinked with the DVS to form a gel;
  • the invention relates to a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with divinylsulfone (DVS), which is sufficiently homogenous to be injected from a 1 ml syringe through a 27G 1 /2 needle over a distance of 55 mm at a speed of 12.5 mm/min with a stable injection force, which after the initial 40 seconds of the injection and until the syringe is empty, varies no more than about 5 Newton (N), preferably no more than about 4 N, more preferably 3 N, 2 N, or most preferably no more than about 1 N.
  • N 5 Newton
  • the invention in a third aspect, relates to a composition
  • a composition comprising a hydrogel as defined in the second aspect, and an active ingredient, preferably the active ingredient is a pharmacologically active agent.
  • a fourth aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a hydrogel as defined in the second aspect, together with a pharmaceutically acceptable carrier, excipient or diluent.
  • a fifth aspect relates to a pharmaceutical composition comprising an effective amount of a hydrogel as defined in the second aspect as a vehicle, together with a pharmacologically active agent.
  • a sixth aspect relates to a cosmetic article comprising as an active ingredient an effective amount of a hydrogel as defined in the second aspect or a composition as defined in any of the third, fourth, or fifth aspects.
  • the invention relates to a sanitary, medical or surgical article comprising a hydrogel as defined in the second aspect or a composition as defined in any of the third, fourth, or fifth aspects, preferably the article is a diaper, a sanitary towel, a surgical sponge, a wound healing sponge, or a part comprised in a band aid or other wound dressing material.
  • An important aspect relates to a medicament capsule or microcapsule comprising a hydrogel as defined in the second aspect or a composition as defined in any of the third, fourth, or fifth aspects.
  • a number of aspects relate to uses of a hydrogel as defined in the second aspect or a composition as defined in any of the third, fourth, or fifth aspects, for the manufacture of a medicament for the treatment of osteoarthritis, cancer, the manufacture of a medicament for an ophtalmological treatment, the manufacture of a medicament for the treatment of a wound, the manufacture of a medicament for angiogenesis, the manufacture of a medicament for the treatment of hair loss or baldness, the manufacture of a moisturizer or a cosmetic, or in a cosmetic treatment.
  • hyaluronic acid is used in literature to mean acidic polysaccharides with different molecular weights constituted by residues of D-glucuronic and N-acetyl-D- glucosamine acids, which occur naturally in cell surfaces, in the basic extracellular substances of the connective tissue of vertebrates, in the synovial fluid of the joints, in the endobulbar fluid of the eye, in human umbilical cord tissue and in cocks' combs.
  • hyaluronic acid is in fact usually used as meaning a whole series of polysaccharides with alternating residues of D-glucuronic and N-acetyl-D-glucosamine acids with varying molecular weights or even the degraded fractions of the same, and it would therefore seem more correct to use the plural term of "hyaluronic acids".
  • the singular term will, however, be used all the same in this description; in addition, the abbreviation "HA" will frequently be used in place of this collective term.
  • Hyaluronic acid is defined herein as an unsulphated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid (GIcUA) linked together by alternating beta-1 ,4 and beta-1 ,3 glycosidic bonds.
  • Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA.
  • hyaluronan and hyaluronic acid are used interchangeably herein.
  • Rooster combs are a significant commercial source for hyaluronan. Microorganisms are an alternative source.
  • U.S. Patent No. 4,801 ,539 discloses a fermentation method for preparing hyaluronic acid involving a strain of Streptococcus zooepidemicus with reported yields of about 3.6 g of hyaluronic acid per liter.
  • European Patent No. EP0694616 discloses fermentation processes using an improved strain of Streptococcus zooepidemicus with reported yields of about 3.5 g of hyaluronic acid per liter.
  • hyaluronic acid or salts thereof may be recombinantly produced, e.g., in a Gram-positive Bacillus host.
  • Hyaluronan synthases have been described from vertebrates, bacterial pathogens, and algal viruses (DeAngelis, P. L., 1999, Cell. MoI. Life Sci. 56: 670-682).
  • WO 99/23227 discloses a Group I hyaluronate synthase from Streptococcus equisimilis.
  • WO 99/51265 and WO 00/27437 describe a Group Il hyaluronate synthase from Pasturella multocida. Ferretti et al.
  • WO 99/51265 describes a nucleic acid segment having a coding region for a Streptococcus equisimilis hyaluronan synthase.
  • the hyaluronan of a recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture medium.
  • the Bacillus cells and cellular debris are physically removed from the culture medium.
  • the culture medium may be diluted first, if desired, to reduce the viscosity of the medium.
  • Many methods are known to those skilled in the art for removing cells from culture medium, such as centrifugation or microfiltration. If desired, the remaining supernatant may then be filtered, such as by ultrafiltration, to concentrate and remove small molecule contaminants from the hyaluronan.
  • a simple precipitation of the hyaluronan from the medium is performed by known mechanisms.
  • Salt, alcohol, or combinations of salt and alcohol may be used to precipitate the hyaluronan from the filtrate.
  • the hyaluronan can be easily isolated from the solution by physical means.
  • the hyaluronan may be dried or concentrated from the filtrate solution by using evaporative techniques known to the art, such as lyophilization or spraydrying.
  • a preferred embodiment relates to the method of the first aspect, wherein the hyaluronic acid or salt thereof is recombinantly produced, preferably by a Gram-positive bacterium or host cell, more preferably by a bacterium of the genus Bacillus.
  • the host cell may be any Bacillus cell suitable for recombinant production of hyaluronic acid.
  • the Bacillus host cell may be a wild-type Bacillus cell or a mutant thereof.
  • Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus agaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. Mutant Bacillus subtilis cells particularly adapted for recombinant expression are described in WO 98/22598.
  • Non- encapsulating Bacillus cells are particularly useful in the present invention.
  • the Bacillus host cell is a Bacillus amyloliquefaciens, Bacillus cla ⁇ sii, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell.
  • the Bacillus cell is a Bacillus amyloliquefaciens cell.
  • the Bacillus cell is a Bacillus clausii cell.
  • the Bacillus cell is a Bacillus lentus cell.
  • the Bacillus cell is a Bacillus licheniformis cell.
  • the Bacillus cell is a Bacillus subtilis cell.
  • the Bacillus host cell is Bacillus subtilis A164 ⁇ 5 (see U.S. Patent No. 5,891,701) or Bacillus subtilis 168 ⁇ 4.
  • the content of hyaluronic acid may be determined according to the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, the average molecular weight of the hyaluronic acid may be determined using standard methods in the art, such as those described by Ueno et a/., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, "Light Scattering University DAWN Course Manual” and "DAWN EOS Manual” Wyatt Technology Corporation, Santa Barbara, California.
  • the hyaluronic acid, or salt thereof, of the present invention has a molecular weight of about 10,000 to about 10,000,000 Da. In a more preferred embodiment it has a molecular weight of about 25,000 to about 5,000,000 Da. In a most preferred embodiment, the hyaluronic acid has a molecular weight of about 50,000 to about 3,000,000 Da.
  • the hyaluronic acid or salt thereof has a molecular weight in the range of between 300,000 and 3,000,000; preferably in the range of between 400,000 and 2,500,000; more preferably in the range of between 500,000 and 2,000,000; and most preferably in the range of between 600,000 and 1 ,800,000.
  • the hyaluronic acid or salt thereof has a low average molecular weight in the range of between 10,000 and 800,000 Da; preferably in the range of between 20,000 and 600,000 Da; more preferably in the range of between 30,000 and 500,000 Da; even more preferably in the range of between 40,000 and 400,000 Da; and most preferably in the range of between 50,000 and 300,000 Da.
  • a preferred embodiment relates to a method of the first aspect, which comprises an inorganic salt of hyaluronic acid, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate.
  • hyaluronic acid preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate.
  • hyaluronic acid preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyal
  • the product produced by the method of the invention may also comprise other ingredients, preferably one or more active ingredient, preferably one or more pharmacologically active substance, and also preferably a water-soluble excipient, such as lactose or a non-biologically derived sugar.
  • Non-limiting examples of an active ingredient or pharmacologically active substance which may be used in the present invention include vitamin(s), protein and/or peptide drugs, such as, human growth hormone, bovine growth hormone, porcine growth hormone, growth homorne releasing hormone/peptide, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, macrophage-colony stimulating factor, erythropoietin, bone morphogenic protein, interferon or derivative thereof, insulin or derivative thereof, atriopeptin-lll, monoclonal antibody, tumor necrosis factor, macrophage activating factor, interleukin, tumor degenerating factor, insulin-like growth factor, epidermal growth factor, tissue plasminogen activator, factor MV, factor IMV, and urokinase.
  • vitamin(s) such as, human growth hormone, bovine growth hormone, porcine growth hormone, growth homorne releasing hormone/peptide, granulocyte-colony stimulating factor,
  • a water-soluble excipient may be included for the purpose of stabilizing the active ingredient(s), such excipient may include a protein, e.g., albumin or gelatin; an amino acid, such as glycine, alanine, glutamic acid, arginine, lysine and a salt thereof; carbohydrate such as glucose, lactose, xylose, galactose, fructose, maltose, saccharose, dextran, mannitol, sorbitol, trehalose and chondroitin sulphate; an inorganic salt such as phosphate; a surfactant such as TWEEN® (ICI), poly ethylene glycol, and a mixture thereof.
  • the excipient or stabilizer may be used in an amount ranging from 0.001 to 99% by weight of the product.
  • compositions and pharmaceuticals comprising, among other constituents, an effective amount of the crosslinked HA product, and an active ingredient, preferably the active ingredient is a pharmacologically active agent; a pharmaceutically acceptable carrier, excipient or diluent, preferably a water-soluble excipient, and most preferably lactose.
  • a preferred embodiment of the invention relates to products or compositions of the invention comprised in an effervescent tablet, which may otherwise be formulated as described in the art.
  • an effervescent tablet may comprise citric acid, sodium bicarbonate, and an oligosaccharide or other sugar.
  • Effervescent tablets are easy to store, and with the fast-dissolving product of the present invention, they are quickly dissolved and thus provide an ideal means of oral administration.
  • aspects of the invention relate to articles comprising a product as defined in the first aspect or a composition as defined in the aspects and embodiments above, e.g., a cosmetic article, a sanitary article, a medical or surgical article.
  • the invention relates to a medicament capsule or microcapsule comprising a product as defined in the first aspect or a composition as defined in other aspects and embodiments of the invention.
  • Figure 1 illustrates the weight loss of DVS-HA hydrogels resulting from Hyaluronidase degradation as a function of time.
  • DVS-HA hydrogels prepared with a heating step ('Heated'), as described in Example 2 were compared to DVS-HA hydrogels which had not been heat treated ('Not heated”).
  • Figure 3 shows the elastic modulus (G'), labelled with a cirle, and the shear loss or viscous modulus (G"), labelled with a square, of two HA hydrogels prepared according to the invention, one prepared with a HA/DVS ratio of 10:1 and 6% HA, and the other with a HA/DVS ratio of 15:1 and 6% HA, as described in detail in example 7 below.
  • G' elastic modulus
  • G shear loss or viscous modulus
  • the elastic modulus (G': circle) of the HA/DVS 10:1 hydrogel is the upper line (y-axis) at all frequencies (x-axis), and the shear loss modulus (G": square) of the HA/DVS 10:1 hydrogel is the lower line, except at the extreme left-hand side of the x-axis, where it is the upper line.
  • Figure 4 shows the syringeability of DVS cross-linked HA hydrogels (HA/DVS 10:1 , wt) prepared following the process described in example 2 herein ('new, heated'), and a hydrogel prepared according to the prior art (see US patent 4,582,865, example 1) without heating, as described in example 9 below.
  • the y-axis shows the injection force in Newton, begining at 0.0 with increments of 2.5, and ending at 35.0.
  • Figure 5 shows the syringeability of DVS cross-linked HA hydrogels (HA/DVS 15:1 , wt) prepared following the process described in example 2 herein ('new, heated'), and a hydrogel prepared according to the prior art (see US patent 4, 582,865, example 1) without heating, as described in example 9 below.
  • the y-axis shows the injection force in Newton, begining at 0.0 with increments of 2.5,and ending at 30.0.
  • the first aspect of the invention relates to a method of producing a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with divinylsulfone (DVS), said method comprising the steps of:
  • step (b) adding DVS to the solution of step (a), whereby the hyaluronic acid, or salt thereof, is crosslinked with the DVS to form a gel; (c) treating the gel of step (b) with a buffer, wherein the gel swells and forms a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with DVS.
  • a buffer wherein the gel swells and forms a hydrogel comprising hyaluronic acid, or salt thereof, crosslinked with DVS.
  • the invention relates to the method of the first aspect, wherein the hyaluronic acid, or salt thereof, is recombinantly produced in a Bacillus host cell.
  • a preferred embodiment of the invention relates to a method of the first aspect, wherein the hyaluronic acid, or salt thereof, has an average molecular weight of between 100 and 3,000 kDa, preferably between 500 and 2,000 kDa, and most preferably between 700 and 1 ,80O kDa.
  • the initical concentration of hyaluronic acid, or a salt thereof, in the method of the invention influences the properties of the resulting crosslinked gel, and of the swollen hydrogel.
  • a preferred embodiment of the invention relates to a method of the first aspect, wherein the alkaline solution comprises dissolved hyaluronic acid, or salt thereof, in a concentration of between 0.1% - 40% (w/v).
  • the pH value during the crosslinking reaction also influences the outcome, so in a preferred embodiment the invention relates to a method of the first aspect, wherein the alkaline solution comprises dissolved sodium hydroxide in a concentration of between 0.001 - 2.0 M.
  • a preferred embodiment of the invention relates to a method of the first aspect, wherein DVS is added to the solution of step (a) in a weight ratio of between 1 :1 and 100:1 of HA/DVS (dry weight), preferably between 2:1 and 50:1 of HA/DVS (dry weight).
  • a preferred embodiment of the invention relates to a method of the first aspect, wherein DVS is added with stirring to the solution of step (a), and wherein the solution temperature is maintained in the range of 5 0 C - 5O 0 C, preferably in the range of 15 0 C - 4O 0 C, more preferably in the range of 2O 0 C - 30 0 C; preferably the stirring is continued for a period of between 1 - 180 minutes.
  • the DVS is added without stirring to the solution of step (a).
  • a preferred embodiment of the invention relates to a method of the first aspect, wherein the solution temperature in step (b) is heated to a temperature in the range of 2O 0 C - 100 0 C, preferably in the range of 25 0 C - 8O 0 C, more preferably in the range of 3O 0 C - 6O 0 C 1 and most preferably in the range of 35 0 C - 55 0 C, and wherein the temperature is maintained in this range for a period of at least 5 minutes, preferably at least 10 minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or most preferably at least 180 minutes; preferably without stirring.
  • the gel is maintained for a period of at least 5 minutes, preferably at least 10 minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or most preferably at least 180 minutes, at a temperature in the range of O 0 C - 4O 0 C, preferably in the range of 1O 0 C - 3O 0 C.
  • the buffer comprises a buffer with a pH value in the range of 2.0 - 8.0, preferably in the range of 5.0 - 7.5.
  • a suitable buffer is chosen with a pH value, which results in that the swollen hydrogel has a pH value as close to neutral as possible.
  • the buffer comprises a buffer with a pH value, which results in that the hydrogel has a pH value between 5.0 and 7.5.
  • the buffer in the method of the first aspect comprises a phosphate buffer and/or a saline buffer.
  • the buffer In the swelling step the buffer must have a sufficient volume for it to accommodate the swelling gel until the gel is fully swollen. Accordingly, in a preferred embodiment of the method of the first aspect, the buffer in step (c) has a volume of at least 3 times the volume of the gel of step (b).
  • the swelling in step (c) is carried out at a temperature of between 2O 0 C - 5O 0 C for a period of at least 5 minutes, preferably at least 10 minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or most preferably at least 180 minutes.
  • the hydrogel formed in step (c) is washed at least once with water, water and a phosphate buffer, water and a saline buffer, or water and a phosphate buffer and a saline buffer, with a pH value in the range of of 2.0 - 8.0, preferably in the range of 5.0 - 7.5.
  • This example illustrates the preparation of DVS-cross-linked HA hydrogels with concomitant swelling and pH adjustment.
  • HA Sodium hyaluronate
  • 0.2M NaOH a 4% (w/v) solution
  • HA/DVS Divinylsulfone
  • the mixtures were stirred at room temperature for 5 min and then allowed to stand at room temperature for 1 h.
  • the gels were then swollen in 160 mL phosphate buffer (pH 4.5 or 6.5) for 24 h, as indicated in Table 1.
  • the pH of the gels was stabilized during the swelling step. After swelling, any excess buffer was removed by filtration and the hydrogels were briefly homogenized with an IKA ® ULTRA-TURRAX ® T25 homogenizer (Ika Labortechnik, DE). The volume and pH of the gels were measured (see Table 2).
  • the pH of the hydrogels ranged from 7.1 to 7.6 (table 2), which confirms that the swelling step can be utilized to adjust the pH in this process. All the hydrogels occupied a volume of 70 mL, which corresponds to a HA concentration of ca. 1.4% (w/v). They were transparent, coherent and homogenous. Softness increased with decreasing cross-linking degree (Table 2).
  • Example 2 Preparation of homogenous DVS-crosslinked HA hydroqels
  • This example illustrates the preparation of highly homogenous DVS-cross-linked HA hydrogels.
  • Sodium hyaluronate (770 kDa, 2 g) was dissolved into 0.2M NaOH with stirring for approx. 1 hour at room temperature to give a 8% (w/v) solution. DVS was then added so that the HA/DVS weight ratio was 7:1. After stirring at room temperature for 5 min, one of the samples was heat treated at 5O 0 C for 2 h without stirring, and then allowed to stand at room temperature overnight. The resulting cross-linked gel was swollen into 200 ml phosphate buffer (pH 5.5) 37 0 C for 42 or 55 h, and finally washed twice with 100 ml water, which was discarded. Volume and pH were measured, as well as the pressure force necessary to push the gels through a 27G * V 2 injection needle (see Table 3).
  • the cross-linked HA hydrogel prepared according to this example exhibited a higher swelling ratio and an increased softness compared to a control hydrogel which was not heat treated (Table 3).
  • the pressure force applied during injection through a 27G *1 /£ needle was more stable than that of the latter sample, indicating that the cross-linked HA hydrogel is more homogenous.
  • This example illustrates the in vitro biostability of DVS-cross-linked HA hydrogels using enzymatic degradation.
  • a bovine testes hyaluronidase (HAase) solution (100 U/mL) was prepared in 30 mM citric acid, 150 mM Na 2 HPO 4 , and 150 mM NaCI (pH 6.3).
  • DVS-HA cross-linked hydrogel samples (ca. 1 mL) were placed into safe-lock glass vials, freeze-dried, and weighed (W 0 ; Formula 1).
  • the enzyme solution (4 mL, 400 U) was then added to each sample and the vials were incubated at 37 0 C under gentle shaking (100-200 rpm).
  • the supernatant was removed and the samples were washed thoroughly with distilled water to remove residual salts, they were then freeze-dried, and finally weighed (W t ; Formula 1).
  • the biodegradation is expressed as the ratio of weight loss to the initial weight of the sample (Formula 1). Weight loss was calculated from the decrease of weight of each sample before and after the enzymatic degradation test. Each biodegradation experiment was repeated three times.
  • DVS-crosslinked HA hydrogels were formulated into creams and serums, that when applied to the skin increase the skin moisturization and elasticity, and provide immediate anti-aging effect, as well as film-forming effect
  • phase E A typical formulation of a water-in-oil (w/o) emulsion containing 2% DVS-cross-linked HA.
  • Each phase (A to E) was prepared separately by mixing the defined ingredients (see Table 4).
  • Phase B was then added to phase A under stirring with a mechanical propel stirring device and at a temperature less than 4O 0 C.
  • Phase C was then added followed by phase D and finally phase E under stirring.
  • Formulations were also made, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, in Phase D, to give a range of w/o formulations.
  • phase E Another typical formulation of a w/o-emulsion containing 2% DVS-crosslinked HA is shown in table 5.
  • Each phase (A to F) in table 5 was prepared separately by mixing the defined ingredients (see Table 5).
  • Phase B was mixed with phase A and the resulting oil phase was heated at 75 0 C.
  • Phase C was also heated to 75 0 C.
  • the oil phase was added to phase C at 75 0 C under stirring with a mechanical propel stirring device.
  • the emulsion was then cooled down to less than 4O 0 C 1 after which phase D was added, followed by phase E and finally phase F under stirring.
  • Formulations were also made, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, in Phase E, to give a range of w/o formulations.
  • a typical formulation of a silicone serum containing 2% DVS-cross-linked HA was prepared as shown in table 6. All ingredients were mixed at the same time under very high stirring and at less than 4O 0 C (see table 6). Formulations were also prepared, wherein the HA hydrogel concentration was 4%, 6% and 8%, respectively, to give a range of serums.
  • DVS cross-linked HA hydrogels with neutral pH are obtained after swelling in phosphate buffer (pH 7.0) for 8 to 14 hours, depending on the degree of cross-linking.
  • a set of DVS cross-linked HA hydrogels was prepared as described in the above, using from 4 to 8% HA solution, and using various amounts of DVS cross-linker, as indicated in Table 7.
  • the decrease in pH is shown for the HA 6% solution and two different ratios of HA/DVS in figure 2, where the HA/DVS ratio of 10:1 is labelled with triangles, and 15:1 is labelled with squares. In these two cases, pH was neutralized within 8 hours. In contrast, neutral pH was reached after 14 hour-swelling for hydrogels with either a higher HA concentration (e.gr. 8%) or a higher degree of cross-linking (e.g. HA/DVS ratio of 2.5).
  • Example 7 Visco elastic properties of hvdrogels based on DVS-crosslinked HA
  • Theological measurements were performed on a Physica MCR 301 rheometer (Anton Paar, Ostfildern, Germany) using a plate-plate geometry and at a controlled temperature of 25°C.
  • the visco-elastic behavior of the samples was investigated by dynamic amplitude shear oscillatory tests, in which the material was subjected to a sinusoidal shear strain.
  • strain/amplitude sweep experiments were performed to evaluate the region of deformation in which the linear viscoelasticity is valid.
  • the strain typically ranged from 0.01 to
  • the shear storage modulus (or elastic modulus G 1 ) and the shear loss modulus (or viscous modulus, G") values were recorded from frequency sweep experiments at a constant shear strain (10%) and at a frequency between 0.1 and 10 Hz.
  • the geometry, the NF and the gap were PP 25, 2 and 1 mm, respectively.
  • G 1 gives information about the elasticity or the energy stored in the material during deformation
  • G" describes the viscous character or the energy dissipated as heat.
  • the elastic modulus gives information about the capability of the sample to sustain load and return in the initial configuration after an imposed stress or deformation. In all experiments, each sample was measured at least three times. The results ( Figure 3) showed that for both hydrogels:
  • G' is ALMOST independent of the frequency.
  • G' is one order of magnitude higher than G"
  • the overall rheological response is due to the contributions of physical and chemical crosslinks, and to topological interactions among the HA macromolecules.
  • the interactions among the chains bring about a reduction of their intrinsic mobility that is not able to release stress, and consequently the material behaves as a three- dimensional network, where the principal mode of accommodation of the applied stress is by network deformation.
  • this hydrogel was more elastic than that with a lower degree of cross-linking (i.e. higher ratio of HA/DVS: 15:1). Indeed, the higher the number of permanent covalent cross-links, the larger the number of entanglements, and therefore the higher the elastic response of the hydrogel.
  • the hydrogels were subjected to periodic oscillation in a dynamic experiment (small amplitude frequency sweep tests) to evaluate the dependence of the elastic and viscous moduli, G' and G".
  • the frequency range was 0.01 Hz - 10 Hz.
  • preliminary strain sweep tests were performed on the samples at the oscillation frequency of 1 Hz. The tests were repeated at least three times on each sample.
  • the values of the elastic modulus can be used to estimate the parameter of the network structure. As G is proportional to the number of entanglements (Ferry, 1980), the elastic modulus can be expressed through:
  • RT is the thermal energy
  • z is the number of the entanglement points or cross-linking point expressed in mol/volume.
  • the parameter z can be calculated by:
  • M e is the average molecular weight of the polymer segments between two entanglements.
  • A is Avogadro's number.
  • Example 9 Syringeability of DVS-crosslinked HA hydrogels
  • the syringeability of DVS cross-linked HA hydrogels prepared according to the present invention was compared to that of hydrogels prepared according to prior art, e.g., as in example 1 of US Patent No 4,582,865.
  • the syringeability was measured on a Texture analyzer (Stable Micro Systems, TA. XT Plus) as the force (in N) needed to inject the hydrogel through a 27GYz needle over a distance of 55 mm at a speed of 12.5 mm/min. Hydrogel samples were transferred into a 1 mL-syringe fitted with a 27G/4 needle and the syringe was placed in the holder. Each sample was ⁇ measured three times.
  • Figures 4 and 5 illustrate the syringeability of DVS cross-linked HA hydrogels with HA/DVS weight ratios of 10:1 and 15:1 , respectively.
  • the injection profiles recorded in figures 4 and 5 are characteristic of the sample homogeneity. Indeed, the more stable the applied injection force is, the more homogenous the hydrogel is. Moreover, a low force corresponds to an easy injection of the hydrogel by the operator.
  • cross-linked hydrogels prepared according to the present invention were easier to inject through a fine needle, as demonstrated by the lower force required. It is noteworthy that the injection force increases with an increasing degree of cross-linking due to the formation of a stronger network.
  • Example 10 Formulations of crosslinked DVS-HA hydrogels for local ophthalmology
  • a typical formulation of a 500 ml_ eye-drop solution containing 1% (w/v) DVS-cross ⁇ linked HA is shown in table 9. All ingredients were weighed and transferred into a 500 mL volumetric flask. Water (300 mL) was added and the mixture was stirred at room temperature for 5 h. pH was adjusted to 7.2 with 2M NaOH and the volume was adjusted to exactly 500 ml_ with milliQ water.
  • a DVS-cross-linked HA hydrogel was prepared using 1.5 g of sodium HA in 0.2 M NaOH to give a 6% (w/v) solution.
  • the HA/DVS weight ratio was 10:1.
  • the hydrogel was prepared in three replicates according to the procedure described in example 2 until the swelling step, after which it was treated as follows: After incubation in an oven at 5O 0 C for two hours, the hydrogel was immersed into Na2HPO4/NaH2PO4 buffer (1 L, 50 mM, pH 7.0) containing the preservative (2-phenoxyethanol/3[(2-ethylhexyl)oxy]1 ,2-propanediol).
  • the concentration of preservative was 10 mL/mL to target a final concentration of 1% (v/v) in the swollen hydrogel. It was anticipated that the preservative would diffuse into the hydrogel during the incubation, and that at the same time, microbial contamination in the buffer would be prevented.
  • the vessel was covered with parafilm and placed in an oven at 37 0 C. After 1 h, the swelling bath was removed and the hydrogel was swollen in a fresh phosphate buffer containing 10 mL/mL preservative for 6-7 h. This step was repeated until the swelling time was 12 h, whereafter the pH was measured. Swelling was continued for another 2.5 h to reach neutral pH.
  • the amount of preservative incorporated into the hydrogel was determined by UV- spectrophotometry (Thermo Electron, Nicolet, Evolution 900, equipment nr. 246-90). A 1% (v/v) solution of the preservative in phosphate buffer was first analyzed to select the wavelength. Approximately 5 mL of hydrogel were collected using a pipette. Typically, sam ⁇ ples were collected in the center of the swollen round hydrogel, and in the north, east, south, and west "sides" of the round gel. The samples were then transferred into a cuvette and the absorbance was read at 292 nm. Each sample was read three times and the absorbance was zeroed against a blank DVS- cross-linked HA hydrogel, containing no preservative.

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Abstract

L'invention concerne des méthodes permettant de produire un hydrogel homogène comprenant de l'acide hyaluronique, ou un sel de ce dernier, réticulé avec du divinylsulfone (DVS). Cette méthode consiste : (a) à préparer une solution alcaline d'acide hyaluronique ou d'un sel de ce dernier ; (b) à ajouter le DVS à la solution de l'étape (a), ce qui entraîne la réticulation de l'acide hyaluronique, ou d'un sel de ce dernier, avec le DVS de façon à former un gel ; (c) à traiter le gel de l'étape (b) avec un tampon, ce qui fait gonfler le gel, lequel forme un hydrogel comprenant de l'acide hyaluronique, ou un sel de ce dernier, réticulé avec le DVS.
PCT/DK2005/000753 2004-11-24 2005-11-24 Methode permettant de reticuler de l'acide hyaluronique avec du divinylsulfone WO2006056204A1 (fr)

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CN2005800471013A CN101107270B (zh) 2004-11-24 2005-11-24 用二乙烯基砜交联透明质酸的方法
US11/719,790 US8481080B2 (en) 2004-11-24 2005-11-24 Method of cross-linking hyaluronic acid with divinulsulfone
PL05804061T PL1817347T3 (pl) 2004-11-24 2005-11-24 Sposób sieciowania kwasu hialuronowego za pomocą diwinylosulfonu
EP05804061.9A EP1817347B1 (fr) 2004-11-24 2005-11-24 Methode permettant de reticuler de l'acide hyaluronique avec de la divinylsulfone
DK05804061.9T DK1817347T3 (en) 2004-11-24 2005-11-24 Process for Crosslinking Hyaluronic Acid with Divinyl Sulfone
ES05804061.9T ES2636998T3 (es) 2004-11-24 2005-11-24 Método de reticulación de ácido hialurónico con divinil sulfona
US13/912,894 US20130338100A1 (en) 2004-11-24 2013-06-07 Method of Cross-Linking Hyaluronic Acid with Divinylsulfone
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Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008126803A1 (fr) * 2007-04-06 2008-10-23 Shiseido Company, Ltd. Composition ophtalmique comprenant de l'acide hyaluronique réticulé soluble
WO2009077620A1 (fr) * 2007-12-19 2009-06-25 Novozymes Biopharma Dk A/S Acide hyaluronique réticulé en émulsion
EP2199308A1 (fr) * 2007-09-28 2010-06-23 Shiseido Company, Ltd. Poudre d'acide hyaluronique réticulé apte à gonfler et son procédé de production
WO2011014432A1 (fr) * 2009-07-30 2011-02-03 Carbylan Biosurgery, Inc. Compositions de polymère d'acide hyaluronique et procédés associés
WO2012089179A1 (fr) 2010-12-31 2012-07-05 Contipro Biotech S.R.O. Fibres en hyaluronane, procédé pour leur préparation et leur utilisation
WO2013021249A1 (fr) 2011-08-10 2013-02-14 Glycores 2000 S.R.L. Hyaluronate de faible masse moléculaire, réticulé, résistant à la dégradation
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WO2014096257A1 (fr) * 2012-12-19 2014-06-26 Novozymes Biopharma Dk A/S Éponge d'acide hyaluronique réticulé lyophilisée
EP2699274B1 (fr) 2011-04-20 2015-03-04 Carbylan Therapeutics, Inc. Compositions de formation de gel in situ
US20150320873A1 (en) * 2012-11-27 2015-11-12 Contipro Biotech S.R.O C6-C18-Acylated Derivative of Hyaluronic Acid, Method of Preparation Thereof, Nanomicellar Composition on its Basis, Method of Preparation Thereof and Method of Preparation Stabilized Nanomicellar Composition, and Use Thereof
US9353194B2 (en) 2009-05-11 2016-05-31 Teoxane Process for preparing a crosslinked hyaluronic acid gel via homogenization in a deformable pouch
US9393189B2 (en) 2010-12-06 2016-07-19 Teoxane Process of preparing a crosslinked gel
US9403918B2 (en) 2009-12-11 2016-08-02 Contipro Pharma A.S. Oxidized derivative of hyaluronic acid, a method of preparation thereof and a method of modification thereof
US9434791B2 (en) 2009-12-11 2016-09-06 Contipro Pharma A.S. Method of preparation of an oxidized derivative of hyaluronic acid and a method of modification thereof
US9492586B2 (en) 2012-02-28 2016-11-15 Contipro Biotech S.R.O. Derivatives of hyaluronic acid capable of forming hydrogels
US9522966B2 (en) 2012-08-08 2016-12-20 Contipro Biotech S.R.O. Hyaluronic acid derivative, method of preparation thereof, method of modification thereof and use thereof
US9597277B2 (en) 2006-12-22 2017-03-21 Croma-Pharma Gesellschaft M.B.H. Use of polymers
US10023658B2 (en) 2014-03-11 2018-07-17 Contipro A.S. Conjugates of oligomer of hyaluronic acid or of a salt thereof, method of preparation thereof and use thereof
US10307512B2 (en) 2014-12-15 2019-06-04 Teoxane Process for preparing hydrogels
US10414832B2 (en) 2015-06-26 2019-09-17 Contipro A.S Derivatives of sulfated polysaccharides, method of preparation, modification and use thereof
US10434214B2 (en) 2011-09-06 2019-10-08 Allergan, Inc. Hyaluronic acid-collagen matrices for dermal filling and volumizing applications
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US10759878B2 (en) 2015-06-15 2020-09-01 Contipro A.S. Method of crosslinking of polysaccharides using photoremovable protecting groups
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FR2861734B1 (fr) 2003-04-10 2006-04-14 Corneal Ind Reticulation de polysaccharides de faible et forte masse moleculaire; preparation d'hydrogels monophasiques injectables; polysaccharides et hydrogels obtenus
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WO2008147867A2 (fr) 2007-05-23 2008-12-04 Allergan, Inc. Collagène réticulé et utilisations de ce dernier
US20110077737A1 (en) * 2007-07-30 2011-03-31 Allergan, Inc. Tunably Crosslinked Polysaccharide Compositions
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US8394784B2 (en) 2007-11-30 2013-03-12 Allergan, Inc. Polysaccharide gel formulation having multi-stage bioactive agent delivery
US8394782B2 (en) 2007-11-30 2013-03-12 Allergan, Inc. Polysaccharide gel formulation having increased longevity
US8450475B2 (en) 2008-08-04 2013-05-28 Allergan, Inc. Hyaluronic acid-based gels including lidocaine
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US9371402B2 (en) * 2009-04-09 2016-06-21 Scivision Biotech Inc. Method for producing cross-linked hyaluronic acid
US9114188B2 (en) 2010-01-13 2015-08-25 Allergan, Industrie, S.A.S. Stable hydrogel compositions including additives
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US8883139B2 (en) 2010-08-19 2014-11-11 Allergan Inc. Compositions and soft tissue replacement methods
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US9737395B2 (en) 2015-06-26 2017-08-22 Phi Nguyen Systems and methods for reducing scarring
US10130578B2 (en) 2015-07-23 2018-11-20 Johnson & Johnson Consumer Inc. Topical delivery of skin compositions having low pH
EP3328351A1 (fr) 2015-07-27 2018-06-06 Galderma S.A. Procédé de réticulation efficace d'acide hyaluronique
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WO2019215572A1 (fr) * 2018-05-08 2019-11-14 Fidia Farmaceutici S.P.A. Pansement pour le traitement de la peau endommagée
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CN114209869A (zh) * 2022-01-14 2022-03-22 北京极暖网络科技有限公司 自发热玻尿酸材料、制备方法及裤式卫生巾

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000079A1 (fr) * 1984-06-08 1986-01-03 Pharmacia Ab GEL D'ACIDE HYALURONIQUE RETICULE A UTILISER COMME SUBSTITUT DE l'HUMEUR VITREUSE
US4582865A (en) * 1984-12-06 1986-04-15 Biomatrix, Inc. Cross-linked gels of hyaluronic acid and products containing such gels
EP0224987A2 (fr) * 1985-11-29 1987-06-10 Biomatrix, Inc. Systèmes à base de l'hyaluronanne, de ses dérivés et de ses sels pour la libération de médicaments et leur procédé de fabrication
US4713448A (en) * 1985-03-12 1987-12-15 Biomatrix, Inc. Chemically modified hyaluronic acid preparation and method of recovery thereof from animal tissues
WO2005066215A1 (fr) * 2003-12-30 2005-07-21 Genzyme Corporation Gels cohesifs de hyaluronane et/ou hylane reticules, preparation et utilisation
WO2005112888A2 (fr) * 2004-05-20 2005-12-01 Mentor Corporation Procede de preparation d'hydrogels polymeres injectables

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141973A (en) * 1975-10-17 1979-02-27 Biotrics, Inc. Ultrapure hyaluronic acid and the use thereof
US4716224A (en) 1984-05-04 1987-12-29 Seikagaku Kogyo Co. Ltd. Crosslinked hyaluronic acid and its use
JPS6345198A (ja) 1986-04-23 1988-02-26 Sumitomo Electric Ind Ltd 多元系結晶の製造方法
IT1198449B (it) 1986-10-13 1988-12-21 F I D I Farmaceutici Italiani Esteri di alcoli polivalenti di acido ialuronico
US5017229A (en) 1990-06-25 1991-05-21 Genzyme Corporation Water insoluble derivatives of hyaluronic acid
US5356883A (en) 1989-08-01 1994-10-18 Research Foundation Of State University Of N.Y. Water-insoluble derivatives of hyaluronic acid and their methods of preparation and use
US5767079A (en) * 1992-07-08 1998-06-16 Celtrix Pharmaceuticals, Inc. Method of treating ophthalmic disorders using TGF -β
US5866120A (en) * 1995-11-22 1999-02-02 Advanced Corneal Systems, Inc. Method for accelerating clearance of hemorrhagic blood from the vitreous humor with hyaluronidase
IT1303735B1 (it) * 1998-11-11 2001-02-23 Falorni Italia Farmaceutici S Acidi ialuronici reticolati e loro usi medici.
FR2827290B1 (fr) * 2001-07-13 2004-07-09 Pierre Jouan Biotechnologies Sa Procede d'obtention d'une fraction proteique enrichie en tgf-beta sous forme activee, fraction proteique et applications therapeutiques
AU2002366711C1 (en) * 2001-12-21 2009-01-22 Novozymes A/S Methods for producing hyaluronan in a recombinant host cell
US20050222081A1 (en) 2002-04-17 2005-10-06 Gianolio Diego A Cross-linked hyaluronate compounds
EP1592715A1 (fr) 2003-01-31 2005-11-09 Biosphere S.p.A. Gels solubles et biocompatibles d'acide hyalonurique reticule avec des l-acides amines ou des l-aminoesters bifonctionnels

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986000079A1 (fr) * 1984-06-08 1986-01-03 Pharmacia Ab GEL D'ACIDE HYALURONIQUE RETICULE A UTILISER COMME SUBSTITUT DE l'HUMEUR VITREUSE
US4582865A (en) * 1984-12-06 1986-04-15 Biomatrix, Inc. Cross-linked gels of hyaluronic acid and products containing such gels
US4713448A (en) * 1985-03-12 1987-12-15 Biomatrix, Inc. Chemically modified hyaluronic acid preparation and method of recovery thereof from animal tissues
EP0224987A2 (fr) * 1985-11-29 1987-06-10 Biomatrix, Inc. Systèmes à base de l'hyaluronanne, de ses dérivés et de ses sels pour la libération de médicaments et leur procédé de fabrication
WO2005066215A1 (fr) * 2003-12-30 2005-07-21 Genzyme Corporation Gels cohesifs de hyaluronane et/ou hylane reticules, preparation et utilisation
WO2005112888A2 (fr) * 2004-05-20 2005-12-01 Mentor Corporation Procede de preparation d'hydrogels polymeres injectables

Cited By (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9597277B2 (en) 2006-12-22 2017-03-21 Croma-Pharma Gesellschaft M.B.H. Use of polymers
WO2008126803A1 (fr) * 2007-04-06 2008-10-23 Shiseido Company, Ltd. Composition ophtalmique comprenant de l'acide hyaluronique réticulé soluble
EP2199308A4 (fr) * 2007-09-28 2011-02-23 Shiseido Co Ltd Poudre d'acide hyaluronique réticulé apte à gonfler et son procédé de production
US8691957B2 (en) 2007-09-28 2014-04-08 Shiseido Company, Ltd. Swellable crosslinked hyaluronan powder and method for producing the same
EP2199308A1 (fr) * 2007-09-28 2010-06-23 Shiseido Company, Ltd. Poudre d'acide hyaluronique réticulé apte à gonfler et son procédé de production
US20100311963A1 (en) * 2007-12-19 2010-12-09 Novozymes Biopharma Dk A/S Crosslinked-hyaluronic acid in emulsion
US20120258155A1 (en) * 2007-12-19 2012-10-11 Novozymes Biopolymer A/S Crosslinked hyaluronic acid in emulsion
US9109051B2 (en) 2007-12-19 2015-08-18 Evonik Goldschmidt Gmbh Crosslinked hyaluronic acid in emulsion
AU2008337407B2 (en) * 2007-12-19 2013-09-05 Novozymes Biopharma Dk A/S Crosslinked hyaluronic acid in emulsion
WO2009077620A1 (fr) * 2007-12-19 2009-06-25 Novozymes Biopharma Dk A/S Acide hyaluronique réticulé en émulsion
US10463595B2 (en) 2008-09-02 2019-11-05 Allergan Holdings France S.A.S. Threads of hyaluronic acid and/or derivatives thereof, methods of making thereof and uses thereof
US11154484B2 (en) 2008-09-02 2021-10-26 Allergan Holdings France S.A.S. Threads of hyaluronic acid and/or derivatives thereof, methods of making thereof and uses thereof
US9353194B2 (en) 2009-05-11 2016-05-31 Teoxane Process for preparing a crosslinked hyaluronic acid gel via homogenization in a deformable pouch
US8790702B2 (en) 2009-07-30 2014-07-29 Carbylan Therapeutics, Inc. Modified hyaluronic acid polymer compositions and related methods
WO2011014432A1 (fr) * 2009-07-30 2011-02-03 Carbylan Biosurgery, Inc. Compositions de polymère d'acide hyaluronique et procédés associés
US9980977B2 (en) 2009-07-30 2018-05-29 Carbylan Therapeutics, Inc. Modified hyaluronic acid polymer compositions and related methods
JP2013501091A (ja) * 2009-07-30 2013-01-10 カービラン バイオサージェリー， インコーポレイテッド 修飾ヒアルロン酸ポリマー組成物および関連する方法
AU2010276574B2 (en) * 2009-07-30 2015-08-20 Carbylan Therapeutics, Inc. Modified hyaluronic acid polymer compositions and related methods
KR101551681B1 (ko) 2009-07-30 2015-09-09 카르빌란 테라퓨틱스, 인코포레이티드 변형된 히알루론산 폴리머 조성물 및 관련방법
US9192678B2 (en) 2009-07-30 2015-11-24 Carbylan Therapeutics, Inc. Modified hyaluronic acid polymer compositions and related methods
CN102573941A (zh) * 2009-07-30 2012-07-11 卡比兰生物外科公司 改性透明质酸聚合物组合物和相关的方法
US9446136B2 (en) 2009-07-30 2016-09-20 Carbylan Therapeutics, Inc. Modified hyaluronic acid polymer compositions and related methods
US9434791B2 (en) 2009-12-11 2016-09-06 Contipro Pharma A.S. Method of preparation of an oxidized derivative of hyaluronic acid and a method of modification thereof
US9403918B2 (en) 2009-12-11 2016-08-02 Contipro Pharma A.S. Oxidized derivative of hyaluronic acid, a method of preparation thereof and a method of modification thereof
US10806821B2 (en) 2010-01-13 2020-10-20 Allergan Industrie, Sas Heat stable hyaluronic acid compositions for dermatological use
US10905797B2 (en) 2010-03-22 2021-02-02 Allergan, Inc. Polysaccharide and protein-polysaccharide cross-linked hydrogels for soft tissue augmentation
US9393189B2 (en) 2010-12-06 2016-07-19 Teoxane Process of preparing a crosslinked gel
WO2012089179A1 (fr) 2010-12-31 2012-07-05 Contipro Biotech S.R.O. Fibres en hyaluronane, procédé pour leur préparation et leur utilisation
US9492383B2 (en) 2011-04-20 2016-11-15 Carbylan Therapeutics, Inc. Rapid in-situ gel forming compositions prepared via reaction of vinyl sulfone derivatized hyaluronic acid with thiol derivatized polyethylene glycol at alkaline pH
EP2699274B1 (fr) 2011-04-20 2015-03-04 Carbylan Therapeutics, Inc. Compositions de formation de gel in situ
US11083684B2 (en) 2011-06-03 2021-08-10 Allergan Industrie, Sas Dermal filler compositions
US10994049B2 (en) 2011-06-03 2021-05-04 Allergan Industrie, Sas Dermal filler compositions for fine line treatment
US10624988B2 (en) 2011-06-03 2020-04-21 Allergan Industrie, Sas Dermal filler compositions including antioxidants
US11000626B2 (en) 2011-06-03 2021-05-11 Allergan Industrie, Sas Dermal filler compositions including antioxidants
WO2013021249A1 (fr) 2011-08-10 2013-02-14 Glycores 2000 S.R.L. Hyaluronate de faible masse moléculaire, réticulé, résistant à la dégradation
US10434214B2 (en) 2011-09-06 2019-10-08 Allergan, Inc. Hyaluronic acid-collagen matrices for dermal filling and volumizing applications
US11833269B2 (en) 2011-09-06 2023-12-05 Allergan, Inc. Hyaluronic acid-collagen matrices for dermal filling and volumizing applications
US9492586B2 (en) 2012-02-28 2016-11-15 Contipro Biotech S.R.O. Derivatives of hyaluronic acid capable of forming hydrogels
US9522966B2 (en) 2012-08-08 2016-12-20 Contipro Biotech S.R.O. Hyaluronic acid derivative, method of preparation thereof, method of modification thereof and use thereof
US9907739B2 (en) 2012-10-24 2018-03-06 Teoxane Dermal injectable sterile composition
US10307362B2 (en) 2012-10-24 2019-06-04 Teoxane SA Dermal injectable sterile composition
WO2014064633A1 (fr) 2012-10-24 2014-05-01 Teoxane Processus de préparation de gel réticulé
WO2014064632A1 (fr) 2012-10-24 2014-05-01 Teoxane Composition stérile dermo-injectable
US9999678B2 (en) 2012-11-27 2018-06-19 Contipro A.S. C6-C18-acylated derivative of hyaluronic acid and method of preparation thereof
US20150320873A1 (en) * 2012-11-27 2015-11-12 Contipro Biotech S.R.O C6-C18-Acylated Derivative of Hyaluronic Acid, Method of Preparation Thereof, Nanomicellar Composition on its Basis, Method of Preparation Thereof and Method of Preparation Stabilized Nanomicellar Composition, and Use Thereof
WO2014096257A1 (fr) * 2012-12-19 2014-06-26 Novozymes Biopharma Dk A/S Éponge d'acide hyaluronique réticulé lyophilisée
US10023658B2 (en) 2014-03-11 2018-07-17 Contipro A.S. Conjugates of oligomer of hyaluronic acid or of a salt thereof, method of preparation thereof and use thereof
US10617711B2 (en) 2014-06-30 2020-04-14 Contipro A.S. Antitumor composition based on hyaluronic acid and inorganic nanoparticles, method of preparation thereof and use thereof
US10722444B2 (en) 2014-09-30 2020-07-28 Allergan Industrie, Sas Stable hydrogel compositions including additives
US10307512B2 (en) 2014-12-15 2019-06-04 Teoxane Process for preparing hydrogels
US10689464B2 (en) 2015-03-09 2020-06-23 Contipro A.S. Self-supporting, biodegradable film based on hydrophobized hyaluronic acid, method of preparation and use thereof
US10759878B2 (en) 2015-06-15 2020-09-01 Contipro A.S. Method of crosslinking of polysaccharides using photoremovable protecting groups
US10414832B2 (en) 2015-06-26 2019-09-17 Contipro A.S Derivatives of sulfated polysaccharides, method of preparation, modification and use thereof
US10618984B2 (en) 2016-06-27 2020-04-14 Contipro A.S. Unsaturated derivatives of polysaccharides, method of preparation thereof and use thereof
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PT1817347T (pt) 2017-07-21
EP1817347B1 (fr) 2017-05-17
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WO2006056204A8 (fr) 2006-08-24
US20090155362A1 (en) 2009-06-18
DK1817347T3 (en) 2017-08-14
US20130338100A1 (en) 2013-12-19
CN101107270A (zh) 2008-01-16
ES2636998T3 (es) 2017-10-10

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